Naturally occurring antibodies, including bivalent antibodies, exhibit immunoreactivity to a specific epitope on a particular target antigen. Multispecific antibodies, as the name indicates, are antibodies engineered to recognise and bind more than one epitope, potentially on different target antigens of interest.
Naturally occurring antibodies typically include combinations of heavy and light immunoglobulin chains, wherein the antigen binding properties of the molecule are determined by the variable regions or domains of the heavy and light chains i.e. the VH and VL domains, respectively. More specifically, the antigen binding sites of any antibody typically include residues contributed by three complementarity determining regions (CDRs) within each of the VH and VL domains.
Multispecific antibodies differ from naturally occurring antibodies in that they incorporate more than one VH-VL domain pairing such that they can recognise and bind to more than one epitope. Commercially these antibodies are extremely important for their ability to bind more than one target antigen. However, significant difficulties exist in the manufacture and isolation of multispecific antibodies as a result of mispairings between the different heavy chains and light chains incorporated into the same antibody molecule. These mispairings can lead to the inadvertent production of monospecific antibodies or antibodies having non-functional or non-productive antigen binding sites, thereby reducing the yield of the multispecific antibody of interest.
FIG. 1 illustrates the difficulties that can arise in the production of a bispecific antibody exhibiting immunoreactivity for two distinct epitopes. The bispecific antibody as shown (A) includes two distinct heavy chains and two distinct light chains. However, only the correct pairing of these four immunoglobulin chains gives rise to an antibody having the required binding profile i.e. specificity for both target antigens. There are in fact nine other potential combinations that can form from a mixture of the four heavy and light chains shown, which result in bivalent monospecific antibodies (E and H), monovalent monospecific antibodies (B, C, G and J) and non-binding antibodies (D, F and I). This problem becomes worse the more complex the multispecific antibody molecule i.e. the more epitopes or antigens the antibody is intended to bind.
Various attempts have been made to improve multispecific antibody production by addressing the problem of incorrect chain pairing. Several approaches have focussed on engineering antibodies so as to promote the correct pairing between VH-VL domains. For instance, US2010/0254989A1 describes the construction of bispecific cMet-ErbB1 antibodies, where the VH and VL of the individual antibodies are fused genetically via a GlySer linker. An alternative approach uses rat-mouse quadromas for generating bispecific antibodies, where the mouse and the rat antibody predominantly forms the original VH-VL pairings and the bispecific antibody consists of the rat and the mouse Fc (Lindhofer et al., J Immunol. (1995) 155: 1246-1252).
For bispecific antibodies including an Fc domain, researchers have also focussed on introducing mutations into the constant region of the heavy chains to promote the correct heterodimerization of the Fc portion. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the “knobs-into-holes” (KiH) approach which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary “hole” in the other CH3 domain of the paired heavy chain so as to promote correct pairing of heavy chains.
Researchers have also attempted to resolve the problem of achieving correct association of heavy chain and light chain pairs. One approach uses the CrossMab principle (as reviewed in Klein et al.), which involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings. Others have sought to engineer the interfaces between the paired VH-VL domains or paired CH1-CL domains of the heavy and light chains so as to increase the affinity between the heavy chain and its cognate light chain (Lewis et al. Nature Biotechnology (2014) 32: 191-198). Techniques such as those described above that require extensive antibody engineering have met with some success; however, the production of antibodies harbouring specific mutations can be labour intensive and can result in antibodies which are highly immunogenic in humans and/or suffer from a loss of effector function.
An alternative approach to the production of multispecific antibody preparations having the correct antigen specificity has been the development of methods that enrich for antibodies having the correct heavy chain-light chain pairings. For example, Spiess et al. (Nature Biotechnology (2013) 31: 753-758) describe a method for the production of a MET-EGFR bispecific antibody from a co-culture of bacteria expressing two distinct half-antibodies. Methods have also been described wherein the constant region of at least one of the heavy chains of a bispecific antibody is mutated so as to alter its binding affinity for an affinity agent, for example Protein A. This allows correctly paired heavy chain heterodimers to be isolated based on a purification technique that exploits the differential binding of the two heavy chains to an affinity agent (see US2010/0331527, WO2013/136186). The limitation with methods that select for correct heavy chain heterodimerization based on differential binding is that they do not select for antibodies having the correct heavy chain-light chain pairings such that these techniques are typically applied to multispecific antibodies having a shared or common light chain.
International patent application no. PCT/EP2012/071866 (WO2013/064701) addresses the problem of incorrect chain pairing using a method for multispecific antibody isolation based on the use of anti-idiotypic binding agents, in particular anti-idiotypic antibodies. The anti-idiotypic binding agents are employed in a two-step selection method in which a first agent is used to capture antibodies having a VH-VL domain pairing specific for a first antigen and a second agent is subsequently used to capture antibodies also having a second VH-VL domain pairing specific for a second antigen.
The drawback with this method is that the anti-idiotypic binding agents used to isolate the antibody must be specific for each multispecific antibody produced, depending on its antigen binding profile. Therefore, although the principle of the method described in PCT/EP2012/071866 is generally applicable to the isolation of any multispecific antibody, the reagents i.e. the anti-idiotypic binding agents, must be generated in accordance with the specific VH-VL domain pairings of the multispecific antibody to be isolated.